• 1.

    James SL et al., 2017. Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories: a systematic analysis for the Global Burden of Disease Study. Lancet 392: 17891858.

    • Search Google Scholar
    • Export Citation
  • 2.

    Hay SI et al., 2017. Global, regional, and national disability-adjusted life-years (DALYs) for 333 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet 390: 12601344.

    • Search Google Scholar
    • Export Citation
  • 3.

    World Health Organization , 2020. 2030 Targets for Soil-Transmitted Helminthiases Control Programmes. Available at: https://www.who.int/intestinal_worms/resources/9789240000315/en/. Accessed October 12, 2020.

  • 4.

    World Health Organization , 2011. Helminth Control in School-Age Children: A Guide for Managers of Control Programmes. Geneva, Switzerland: WHO. Available at: https://www.who.int/neglected_diseases/resources/9789241548267/en/. Accessed October 12, 2020.

  • 5.

    Inpankaew T, Schar F, Khieu V, Muth S, Dalsgaard A, Marti H, Traub RJ, Odermatt P, 2014. Simple fecal flotation is a superior alternative to guadruple Kato Katz smear examination for the detection of hookworm eggs in human stool. PLoS Negl Trop Dis 8: e3313.

    • Search Google Scholar
    • Export Citation
  • 6.

    Nikolay B, Brooker SJ, Pullan RL, 2014. Sensitivity of diagnostic tests for human soil-transmitted helminth infections: a meta-analysis in the absence of a true gold standard. Int J Parasitol 44: 765774.

    • Search Google Scholar
    • Export Citation
  • 7.

    Clarke NE, Llewellyn S, Traub RJ, McCarthy J, Richardson A, Nery SV, 2018. Quantitative polymerase chain reaction for diagnosis of soil-transmitted helminth infections: a comparison with a flotation-based technique and an investigation of variability in DNA detection. Am J Trop Med Hyg 99: 10331040.

    • Search Google Scholar
    • Export Citation
  • 8.

    Llewellyn S, Inpankaew T, Nery SV, Gray DJ, Verweij JJ, Clements AC, Gomes SJ, Traub R, McCarthy JS, 2016. Application of a multiplex quantitative PCR to assess prevalence and intensity of intestinal parasite infections in a controlled clinical trial. PLoS Negl Trop Dis 10: e0004380.

    • Search Google Scholar
    • Export Citation
  • 9.

    Easton AV et al., 2016. Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: field-based inferences on the impact of mass deworming. Parasit Vectors 9: 38.

    • Search Google Scholar
    • Export Citation
  • 10.

    Mationg MLS et al., 2017. Status of soil-transmitted helminth infections in schoolchildren in Laguna Province, the Philippines: determined by parasitological and molecular diagnostic techniques. PLoS Negl Trop Dis 11: e0006022.

    • Search Google Scholar
    • Export Citation
  • 11.

    Mejia R, Vicuna Y, Broncano N, Sandoval C, Vaca M, Chico M, Cooper PJ, Nutman TB, 2013. A novel, multi-parallel, real-time polymerase chain reaction approach for eight gastrointestinal parasites provides improved diagnostic capabilities to resource-limited at-risk populations. Am J Trop Med Hyg 88: 10411047.

    • Search Google Scholar
    • Export Citation
  • 12.

    Medley GF, Turner HC, Baggaley RF, Holland C, Hollingsworth TD, 2016. The role of more sensitive helminth diagnostics in mass drug administration campaigns: elimination and health impacts. Adv Parasitol 94: 343392.

    • Search Google Scholar
    • Export Citation
  • 13.

    Stracke K, Jex AR, Traub RJ, 2020. Zoonotic ancylostomiasis: an update of a continually neglected zoonosis. Am J Trop Med Hyg 103: 6468.

  • 14.

    Hii SF, Senevirathna D, Llewellyn S, Inpankaew T, Odermatt P, Khieu V, Muth S, McCarthy J, Traub RJ, 2018. Development and evaluation of a multiplex quantitative real-time polymerase chain reaction for hookworm species in human stool. Am J Trop Med Hyg 99: 11861193.

    • Search Google Scholar
    • Export Citation
  • 15.

    Zendejas-Heredia PA, Hii SF, Colella V, Traub RJ, 2021. Comparison of the egg recovery rates and limit of detection for soil-transmitted helminths using the Kato-Katz thick smear, faecal flotation and quantitative real-time PCR in human stool. PLoS Negl Trop Dis 15: e0009395.

    • Search Google Scholar
    • Export Citation
  • 16.

    Basuni M, Muhi J, Othman N, Verweij JJ, Ahmad M, Miswan N, Rahumatullah A, Aziz FA, Zainudin NS, Noordin R, 2011. A pentaplex real-time polymerase chain reaction assay for detection of four species of soil-transmitted helminths. Am J Trop Med Hyg 84: 338343.

    • Search Google Scholar
    • Export Citation
  • 17.

    Liu J et al., 2013. A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens. J Clin Microbiol 51: 472480.

  • 18.

    Verweij JJ, Canales M, Polman K, Ziem J, Brienen EA, Polderman AM, van Lieshout L, 2009. Molecular diagnosis of Strongyloides stercoralis in faecal samples using real-time PCR. Trans R Soc Trop Med Hyg 103: 342346.

    • Search Google Scholar
    • Export Citation
  • 19.

    Lambert SB, Whiley DM, O’Neill NT, Andrews EC, Canavan FM, Bletchly C, Siebert DJ, Sloots TP, Nissen MD, 2008. Comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction. Pediatrics 122: e615e620.

    • Search Google Scholar
    • Export Citation
  • 20.

    Verweij JJ, Brienen EA, Ziem J, Yelifari L, Polderman AM, Van Lieshout L, 2007. Simultaneous detection and quantification of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples using multiplex real-time PCR. Am J Trop Med Hyg 77: 685690.

    • Search Google Scholar
    • Export Citation
  • 21.

    World Health Organization , 2002. Prevention and Control of Schistosomiasis and Soil-Transmitted Helminthiasis: Report of a WHO Expert Committee. Geneva, Switzerland: WHO. Available at: https://apps.who.int/iris/handle/10665/42588. Accessed November 23, 2020.

  • 22.

    Chichino G, Bernuzzi AM, Bruno A, Cevini C, Atzori C, Malfitano A, Scagila M, 1992. Intestinal capillariasis (Capillaria philippinensis) acquired in Indonesia: a case report. Am J Trop Med Hyg 47: 1012.

    • Search Google Scholar
    • Export Citation
  • 23.

    Uga S, Hoa NT, Noda S, Moji K, Cong L, Aoki Y, Rai SK, Fujimaki Y, 2009. Parasite egg contamination of vegetables from a suburban market in Hanoi, Vietnam. Nepal Med Coll J 11: 7578.

    • Search Google Scholar
    • Export Citation
 
 

 

 

 

 

 

 

Comparison between Quantitative Polymerase Chain Reaction and Sodium Nitrate Flotation Microscopy in Diagnosing Soil-Transmitted Helminth Infections

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  • 1 Kirby Institute, University of New South Wales, Sydney, Australia;
  • | 2 University of Melbourne, Parkville, Victoria, Australia;
  • | 3 Menzies School of Health Research, Charles Darwin University, Darwin, Australia;
  • | 4 Royal Darwin Hospital, Darwin, Australia;
  • | 5 Department of Infectious Diseases, Imperial College London, London, United Kingdom;
  • | 6 Department of Communicable Diseases Control, Ministry of Health, Dili, Timor-Leste;
  • | 7 National Health Laboratory of Timor-Leste, Dili, Timor-Leste

ABSTRACT.

There is evolving interest in alternate microscopy techniques and quantitative polymerase chain reaction (qPCR) to evaluate soil-transmitted helminth (STH) burden. Using data from a cross-sectional survey of 540 schoolchildren across six primary schools in three municipalities of Timor-Leste, we compared the performance of microscopy using sodium nitrate flotation (SNF) and qPCR in determining STH prevalence and infection intensity. Prevalence by qPCR was higher than SNF for Ascaris lumbricoides (17.5% versus 11.2%), hookworm (8.3% versus 1.2%), and Trichuris trichiura (4.7% versus 1.6%). Agreement between SNF and qPCR was fair for hookworm (κ = 0.21) and moderate for A. lumbricoides (κ = 0.59) and T. trichiura (κ = 0.44). Moderate or heavy intensity infections were identified in 15.9% of infections detected by SNF, whereas qPCR identified 36.1% as moderate or heavy infections using cycle threshold to eggs per gram conversion formulas. Quantitative PCR is a promising diagnostic technique, though further studies validating infection intensity correlates are required.

INTRODUCTION

Soil-transmitted helminth (STH) infections remain a major global health issue, affecting almost one billion people globally1 and contributing the highest burden of all the neglected tropical diseases.2 Integral to STH control is preventive chemotherapy targeting school-age children in settings where STH prevalence is 20% or higher, according to World Health Organization (WHO) recommendations.3 To evaluate STH burden (prevalence and infection intensity), accurate diagnostic techniques suitable in resource-limited settings are necessary. Interest is evolving in alternate diagnostic techniques to Kato-Katz, which is the WHO standard method for STH diagnosis and quantification,4 including sodium nitrate flotation (SNF), which demonstrates superior sensitivity than Kato-Katz in detecting hookworm eggs and can be done in preserved stool samples, thereby simplifying fieldwork procedures.5 However, there is concern for the poor sensitivity of microscopy in low-prevalence settings and detecting light intensity infections and the inability to differentiate between hookworm species.5,6 Quantitative polymerase chain reaction (qPCR) is a molecular method gaining recognition in STH diagnostics, demonstrating higher sensitivity than microscopy across a range of settings and capacity to specify hookworm species.5,711 This is particularly advantageous following STH control programs where prevalence and infection intensity is expected to be lower12 and to evaluate the burden of zoonotic hookworm species to inform broader control strategies involving animal reservoirs.13 Furthermore, qPCR can be conducted on preserved stool samples. However, although qPCR quantifies helminth DNA, correlation with the traditional estimates of infection intensity obtained by microscopy (eggs per gram [epg]) is not well established, and studies have demonstrated linear correlations between qPCR cycle threshold (Ct) values and microscopy epg (having either one or both values log-transformed) using fecal seeding experiments and field samples.7,8,14,15 However, direct comparisons between SNF and qPCR infection intensity using Ct to epg conversion formulas are yet to be published. This analysis is the first to compare SNF and qPCR in both determining STH prevalence and infection intensity (in epg) using samples collected in a cross-sectional study in Timor-Leste.

MATERIALS AND METHODS

A cross-sectional survey of school-age children across six primary schools in three municipalities of Timor-Leste was conducted in April–May 2019. The study received Human Research Ethics Committee approval from the University of New South Wales (HC190140) and Timor-Leste Ministry of Health (1545MS-INS/DE/X/2019).

Upon collection of each stool sample, two aliquots (3 g each) were placed into separate centrifuge tubes and preserved with 3 mL of 5% potassium dichromate, kept on ice in the field, and refrigerated upon arrival at the National Health Laboratory, Dili, Timor-Leste. A modified SNF analysis of samples (using two coverslip readings, after being atop the centrifuge tube for 5 minutes each) was conducted at the National Health Laboratory (May 2019) following a 2-day training by a senior parasitologist (RT).5 For qPCR analysis, samples were couriered to the University of Melbourne, Australia, and underwent two quadraplex real-time qPCR assays. The first assay was performed to enumerate Ascaris lumbricoides and Trichuris trichiura and detect Strongyloides stercoralis using Equine Herpesvirus-4 (as an internal qPCR control), and the second was performed to enumerate Necator americanus, Ancylostoma duodenale, and Ancylostoma ceylanicum using human 16S mitochondrial rRNA as an internal qPCR and DNA extraction control.15 Nuclease-free water was used as negative control. The sequences of primers and probes and PCR conditions were based on published information8,1420 and unpublished information from P. A. Zendejas-Heredia et al. (Supplemental Table 1). Quantitative PCR assays were performed from December 2019 to January 2020. Strongyloides stercoralis is not included in this analysis because it is not detected by SNF.

Table 1

Species-specific soil-transmitted helminth prevalence as determined by sodium nitrate flotation and quantitative polymerase chain reaction

OrganismPrevalence by SNF (N = 493)Prevalence by qPCR (N = 531)Prevalence by SNF or qPCR (N = 540)
n% (95% CI)n% (95% CI)n% (95% CI)
Ascaris lumbricoides5511.2 (8.4–13.9)9317.5 (14.3–20.8)10419.3 (15.9–22.6)
Hookworm*61.2 (0.3–2.2)448.3 (5.6–10.6)458.3 (6.0–10.7)
 Necator americanus407.5 (5.3–9.8)
 Ancylostoma duodenale10.2 (0–0.6)
 Ancylostoma ceylanicum30.6 (0–1.2)
 Trichuris trichiura81.6 (0.5–2.7)254.7 (2.9–6.5)264.8 (3.0–6.6)
Any STH6713.6 (10.6–16.6)14627.5 (23.7–31.3)15628.9 (25.1–32.7)

CI = confidence interval; qPCR = quantitative polymerase chain reaction; SNF = sodium nitrate flotation.

Hookworm species cannot be determined by microscopy but can be distinguished by qPCR; therefore, only hookworm prevalence is reported for SNF, and the prevalence of specific hookworm species are reported for qPCR.

The prevalence and 95% confidence intervals for A. lumbricoides, hookworm, T. trichiura, and infection with at least one STH was compared using SNF and qPCR. Identifying specific hookworm species is not possible by microscopy; therefore, an individual was deemed positive for hookworm by SNF if one or more hookworm species was detected. Diagnostic agreement between SNF and qPCR was assessed using Cohen’s Kappa agreement statistics (very good, κ > 0.8; good, 0.6 < κ ≤ 0.8; moderate, 0.4 < κ ≤ 0.6; fair, 0.2 < κ ≤ 0.4; poor, κ ≤ 0.2). In the absence of a true gold standard, a “true positive” was considered an infection detected by either method,7 and the sensitivities of SNF and qPCR were estimated by dividing the positives for each method by the “true positive.” Specificity was assumed to be 100%. Infection intensity by SNF was determined by epg and categorized using WHO criteria.21 Infection intensity by qPCR was determined by correlating Ct values with epg through fecal seeding experiments8,14 and subsequently categorized using WHO criteria.21 Samples seeded with known quantities of STH eggs were analyzed by qPCR to develop the following linear regression models: A. lumbricoides Ct = −3.489log10(epg) + 36.97, R2 = 0.9881; N. americanus Ct = −3.641log10(epg) + 35.02, R2 = 0.982; and T. trichiura Ct = −3.288log10(epg) + 36.73, R2 = 0.9812.15 The Spearman’s rank correlation coefficient was calculated to assess associations between SNF and qPCR in determining infection intensity (very strong, ρ ≥ 0.8; strong, 0.6 ≤ ρ < 0.8; moderate, 0.4 ≤ ρ < 0.6; weak, 0.2 ≤ ρ < 0.4; very weak, ρ < 0.2). A P value < 0.05 was considered statistically significant. For participants with multiple SNF or qPCR results (e.g., multiple samples collected, multiple analyses on same sample, or sample mislabeling), if the results were concordant then one result was included; if the results were discordant then the SNF or qPCR results were excluded. Among the 16 participants with multiple SNF results, 10 had concordant results that had one result included. Among the 10 participants with multiple qPCR results, eight had concordant results that had one result included. All analyses were conducted using SAS version 9.4 (SAS, Cary, NC).

RESULTS

Samples were collected from 548 participants. After removing duplicate samples, samples from 540 participants were included in this analysis. Table 1 compares the prevalence of A. lumbricoides, hookworm, T. trichiura, and infection with any STH by SNF and qPCR. Prevalence was lower for SNF compared with qPCR for all species: A. lumbricoides, 11.2% by SNF versus 17.5% by qPCR; hookworm, 1.2% by SNF versus 8.3% by qPCR; T. trichiura, 1.6% by SNF versus 4.7% by qPCR; and any STH, 13.6% by SNF versus 27.5% by qPCR. The qPCR allowed the identification of hookworm species, with N. americanus being most prevalent (7.5%; 95% confidence interval: 5.3–9.8).

Sensitivity and diagnostic agreement were calculated for the 483 participants with SNF and qPCR results. For A. lumbricoides, SNF sensitivity was 58.1% (54/93), and qPCR was 89.2% (83/93); for hookworm, SNF sensitivity was 12.8% (5/39), and qPCR was 100% (39/39); for T. trichiura, SNF sensitivity was 30.4% (7/23), and qPCR was 95.8% (23/24). The diagnostic agreement between SNF and qPCR was fair for hookworm (κ = 0.21; P < 0.001) and moderate for A. lumbricoides (κ = 0.59, P < 0.001) and T. trichiura (κ = 0.44; P < 0.001) (Table 2).

Table 2

Diagnostic agreement between sodium nitrate flotation and quantitative polymerase chain reaction

SNF resultqPCR detected (n)qPCR not detected (n)Agreement (%)Kappa statistic*P value
Ascaris lumbricoidesDetected4410428 (89.7)0.59< 0.001
Not detected39384
HookwormDetected50444 (92.9)0.21< 0.001
Not detected34439
Trichuris trichiuraDetected71461 (96.4)0.44< 0.001
Not detected16454

qPCR = quantitative polymerase chain reaction; SNF = sodium nitrate flotation.

Kappa agreement classification: < 0.20 = poor; 0.21–0.40 = fair; 0.41–0.60 = moderate; 0.61–0.80 = good; 0.81–1.00 = very good.

Table 3 shows infection intensity and mean epg values by SNF and qPCR (using Ct to epg conversion formulas). For A. lumbrocoides the mean epg by SNF was 2,778 (range: 2–21,654), and by qPCR it was 57,521 (range: 6–1,707,831); for hookworm the mean epg by SNF was 294 (range: 10–1,294). For N. americanus the mean epg by qPCR was 5,898 (range: 3–217,680); for T. trichiura the mean epg by SNF was 81 (range: 2–411) and by qPCR was 282,741 (range: 1–7,065,590). All hookworm and T. trichiura infections were light intensity by SNF, compared with 2/40 (5%) of N. americanus infections and 2/25 (8%) of T. trichiura infections being moderate or heavy intensity by qPCR. Only 11/55 (20%) of A. lumbricoides infections were moderate or heavy intensity by SNF, compared with 53/93 (57.0%) by qPCR. Overall, moderate or heavy infection intensity was identified by SNF in 11/69 (15.9%) cases, whereas 57/158 (36.1%) were moderate or heavy infection intensity by qPCR. The correlation between SNF and qPCR for A. lumbricoides infection intensity was not significant (ρ = 0.20; P = 0.20) and could not be calculated for N. americanus or T. trichiura due to the low number detected by SNF.

Table 3

Infection intensity as determined by sodium nitrate flotation and quantitative polymerase chain reaction*

OrganismLight, n (%)Moderate, n (%)Heavy, n (%)Mean epg (range)
SNF (N = 493)qPCR (N = 531)SNF (N = 493)qPCR (N = 531)SNF (N = 493)qPCR (N = 531)SNFqPCR
Ascaris lumbricoides44 (8.9)40 (7.5)11 (2.2)41 (7.7)012 (2.3)2,778 (2–21,654)57,521 (6–1,707,831)
Hookworm6 (1.2)00294 (10–1,294)
 Necator americanus38 (7.2)1 (0.2)1 (0.2)5,898 (3–217,680)
Trichuris trichiura8 (1.6)23 (4.3)01 (0.2)01 (0.2)81 (2–411)282,741 (1–7,065,590)

epg = eggs per gram; qPCR = quantitative polymerase chain reaction; SNF = sodium nitrate flotation.

Using cycle threshold to epg conversion formulas.

CONCLUSION

This analysis adds to the growing evidence for the value of qPCR in STH diagnostics. Our results demonstrating higher sensitivity of qPCR compared with SNF are consistent with previous analyses.5,7,8,14,15 The lower sensitivity of SNF and diagnostic agreement between SNF and qPCR in our study compared with a previous analysis from Timor-Leste7 may reflect issues with egg recovery using SNF and the operator-dependent nature of microscopy, with the prior study using experienced researchers and this study using routine laboratory technicians. The small number of STH infections detected by SNF, particularly for hookworm and T. trichiura, is one of the main limitations of our study and is likely to have contributed to the poorer diagnostic agreement in our analysis. The small number of STH infections detected by SNF but not qPCR may reflect the heterogeneity of egg distribution within stool samples, false-positive microscopy results due to the presence of eggs from other morphologically similar nematodes,22,23 or the misidentification of fecal material.

Only 15.9% of STH infections detected by SNF were moderate or heavy intensity, whereas 36.1% of infections detected by qPCR were moderate or heavy intensity. Importantly, we did not observe egg embryonation in qPCR samples, which would increase DNA amount beyond that at the time of infection. However, individual sample variations in egg embryonation cannot be excluded if defecation occurred the day prior to sample processing, which would result in falsely elevated qPCR-based egg intensity estimates. Quantitative PCR may also overestimate STH burden by detecting DNA from nonviable eggs and free DNA. There have been difficulties in comparing infection intensity obtained by microscopy, where egg counts are measured directly, and qPCR, where egg counts are estimated based on qPCR Ct values and calibration curves. Previous attempts to derive infection intensity using Ct to epg conversion formulas consistently demonstrate higher infection intensities compared with microscopy, with Hii et al.14 reporting hookworm egg intensity estimates 4- to 8.5-fold higher using Ct conversion formulas compared with field-based microscopy and increasing discrepancy as egg intensities lighten. Additionally, STH egg-seeding experiments demonstrated egg recovery rates of microscopy-based techniques (Kato Katz and SNF) to be 20% lower compared with quadraplex qPCR assays, with qPCR being better able to predict the accuracy of light or moderate egg intensity cut-offs compared with microscopy-based techniques.15 Our study is the first to use Ct to epg conversion formulas to directly compare SNF and qPCR in determining infection intensity using WHO epg thresholds. Additional studies are needed to evolve the application of molecular quantification to determine STH infection intensity.

In conclusion, this analysis further demonstrates the increased sensitivity of qPCR in detecting and quantifying STH infection compared with microscopy. The diagnostic performance of qPCR, in conjunction with straightforward sample preservation, lower potential for interoperator variability, and capacity for species-specific identification of hookworm makes for a promising diagnostic. Where accessible, qPCR should be incorporated into assessments of STH burden to refine quantitative correlations with microscopy techniques and establish validated conversion of qPCR Ct values to epg. This will allow a more accurate assessment of STH burden and evaluation of progress toward STH control and potential elimination.

ACKNOWLEDGMENTS

We thank Clara Guterres and Tessa Wylie (Menzies School of Health Research, Charles Darwin University, Darwin, Australia) for their contributions in study coordination and supporting scientific staff.

REFERENCES

  • 1.

    James SL et al., 2017. Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories: a systematic analysis for the Global Burden of Disease Study. Lancet 392: 17891858.

    • Search Google Scholar
    • Export Citation
  • 2.

    Hay SI et al., 2017. Global, regional, and national disability-adjusted life-years (DALYs) for 333 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet 390: 12601344.

    • Search Google Scholar
    • Export Citation
  • 3.

    World Health Organization , 2020. 2030 Targets for Soil-Transmitted Helminthiases Control Programmes. Available at: https://www.who.int/intestinal_worms/resources/9789240000315/en/. Accessed October 12, 2020.

  • 4.

    World Health Organization , 2011. Helminth Control in School-Age Children: A Guide for Managers of Control Programmes. Geneva, Switzerland: WHO. Available at: https://www.who.int/neglected_diseases/resources/9789241548267/en/. Accessed October 12, 2020.

  • 5.

    Inpankaew T, Schar F, Khieu V, Muth S, Dalsgaard A, Marti H, Traub RJ, Odermatt P, 2014. Simple fecal flotation is a superior alternative to guadruple Kato Katz smear examination for the detection of hookworm eggs in human stool. PLoS Negl Trop Dis 8: e3313.

    • Search Google Scholar
    • Export Citation
  • 6.

    Nikolay B, Brooker SJ, Pullan RL, 2014. Sensitivity of diagnostic tests for human soil-transmitted helminth infections: a meta-analysis in the absence of a true gold standard. Int J Parasitol 44: 765774.

    • Search Google Scholar
    • Export Citation
  • 7.

    Clarke NE, Llewellyn S, Traub RJ, McCarthy J, Richardson A, Nery SV, 2018. Quantitative polymerase chain reaction for diagnosis of soil-transmitted helminth infections: a comparison with a flotation-based technique and an investigation of variability in DNA detection. Am J Trop Med Hyg 99: 10331040.

    • Search Google Scholar
    • Export Citation
  • 8.

    Llewellyn S, Inpankaew T, Nery SV, Gray DJ, Verweij JJ, Clements AC, Gomes SJ, Traub R, McCarthy JS, 2016. Application of a multiplex quantitative PCR to assess prevalence and intensity of intestinal parasite infections in a controlled clinical trial. PLoS Negl Trop Dis 10: e0004380.

    • Search Google Scholar
    • Export Citation
  • 9.

    Easton AV et al., 2016. Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: field-based inferences on the impact of mass deworming. Parasit Vectors 9: 38.

    • Search Google Scholar
    • Export Citation
  • 10.

    Mationg MLS et al., 2017. Status of soil-transmitted helminth infections in schoolchildren in Laguna Province, the Philippines: determined by parasitological and molecular diagnostic techniques. PLoS Negl Trop Dis 11: e0006022.

    • Search Google Scholar
    • Export Citation
  • 11.

    Mejia R, Vicuna Y, Broncano N, Sandoval C, Vaca M, Chico M, Cooper PJ, Nutman TB, 2013. A novel, multi-parallel, real-time polymerase chain reaction approach for eight gastrointestinal parasites provides improved diagnostic capabilities to resource-limited at-risk populations. Am J Trop Med Hyg 88: 10411047.

    • Search Google Scholar
    • Export Citation
  • 12.

    Medley GF, Turner HC, Baggaley RF, Holland C, Hollingsworth TD, 2016. The role of more sensitive helminth diagnostics in mass drug administration campaigns: elimination and health impacts. Adv Parasitol 94: 343392.

    • Search Google Scholar
    • Export Citation
  • 13.

    Stracke K, Jex AR, Traub RJ, 2020. Zoonotic ancylostomiasis: an update of a continually neglected zoonosis. Am J Trop Med Hyg 103: 6468.

  • 14.

    Hii SF, Senevirathna D, Llewellyn S, Inpankaew T, Odermatt P, Khieu V, Muth S, McCarthy J, Traub RJ, 2018. Development and evaluation of a multiplex quantitative real-time polymerase chain reaction for hookworm species in human stool. Am J Trop Med Hyg 99: 11861193.

    • Search Google Scholar
    • Export Citation
  • 15.

    Zendejas-Heredia PA, Hii SF, Colella V, Traub RJ, 2021. Comparison of the egg recovery rates and limit of detection for soil-transmitted helminths using the Kato-Katz thick smear, faecal flotation and quantitative real-time PCR in human stool. PLoS Negl Trop Dis 15: e0009395.

    • Search Google Scholar
    • Export Citation
  • 16.

    Basuni M, Muhi J, Othman N, Verweij JJ, Ahmad M, Miswan N, Rahumatullah A, Aziz FA, Zainudin NS, Noordin R, 2011. A pentaplex real-time polymerase chain reaction assay for detection of four species of soil-transmitted helminths. Am J Trop Med Hyg 84: 338343.

    • Search Google Scholar
    • Export Citation
  • 17.

    Liu J et al., 2013. A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens. J Clin Microbiol 51: 472480.

  • 18.

    Verweij JJ, Canales M, Polman K, Ziem J, Brienen EA, Polderman AM, van Lieshout L, 2009. Molecular diagnosis of Strongyloides stercoralis in faecal samples using real-time PCR. Trans R Soc Trop Med Hyg 103: 342346.

    • Search Google Scholar
    • Export Citation
  • 19.

    Lambert SB, Whiley DM, O’Neill NT, Andrews EC, Canavan FM, Bletchly C, Siebert DJ, Sloots TP, Nissen MD, 2008. Comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction. Pediatrics 122: e615e620.

    • Search Google Scholar
    • Export Citation
  • 20.

    Verweij JJ, Brienen EA, Ziem J, Yelifari L, Polderman AM, Van Lieshout L, 2007. Simultaneous detection and quantification of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples using multiplex real-time PCR. Am J Trop Med Hyg 77: 685690.

    • Search Google Scholar
    • Export Citation
  • 21.

    World Health Organization , 2002. Prevention and Control of Schistosomiasis and Soil-Transmitted Helminthiasis: Report of a WHO Expert Committee. Geneva, Switzerland: WHO. Available at: https://apps.who.int/iris/handle/10665/42588. Accessed November 23, 2020.

  • 22.

    Chichino G, Bernuzzi AM, Bruno A, Cevini C, Atzori C, Malfitano A, Scagila M, 1992. Intestinal capillariasis (Capillaria philippinensis) acquired in Indonesia: a case report. Am J Trop Med Hyg 47: 1012.

    • Search Google Scholar
    • Export Citation
  • 23.

    Uga S, Hoa NT, Noda S, Moji K, Cong L, Aoki Y, Rai SK, Fujimaki Y, 2009. Parasite egg contamination of vegetables from a suburban market in Hanoi, Vietnam. Nepal Med Coll J 11: 7578.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Susana Vaz Nery, Kirby Institute, University of New South Wales, Sydney NSW Australia, 2052. E-mail: snery@kirby.unsw.edu.au

Financial support: This research was funded through a Centres of Research Excellence Grant from the National Health and Medical Research Council, Australia (APP1153727).

Editor’s Note: Due to last minute changes requested by the authors, there are differences between the print and online versions of this manuscript. The online version contains these changes; the print version does not.

Authors’ addresses: Adam W. Bartlett, Naomi E. Clarke, and Susana Vaz Nery, University of New South Wales, Kirby Institute, Sydney, Australia, E-mails: abartlett@kirby.unsw.edu.au, nclarke@kirby.unsw.edu.au, and snery@kirby.unsw.edu.au. Rebecca Traub and Sze Fui Hii, University of Melbourne, Veterinary and Agricultural Sciences, Melbourne, Victoria, Australia, E-mails: rebecca.traub@unimelb.edu.au and sze.hii@unimelb.edu.au. Salvador Amaral and Virginia da Conceicao, Charles Darwin University, Menzies School of Health Research, Darwin, Australia, E-mails: salvador.amaral@menzies.edu.au and virginia.conceicao@menzies.edu.au. Alexander Matthews, Northern Territory Department of Health, Royal Darwin Hospital, Darwin, Australia, E-mail: alextommatt@gmail.com. Patsy A. Zendejas-Heredia, University of Melbourne, Faculty of Veterinary and Agricultural Sciences, Parkville, Victoria, Australia, E-mail: patsy.zendejas@unimelb.edu.au. Paul Arkell, Charles Darwin University, Menzies School of Health Research, Darwin, Australia, and Imperial College London, Department of Infectious Diseases, London, London, United Kingdom, E-mail: paularkell@doctors.org.uk. Merita Antonia Armindo Monteiro, Ministry of Health, Department of Communicable Diseases Control, Dili, Timor-Leste, E-mail: methamonteiro@yahoo.com. Carolina da Costa Maia, Maria Imaculada Soares, and Josefina D. Prisca Guterres, Ministry of Health, National Health Laboratory, Dili, Timor-Leste, E-mails: carolinamaia_26@yahoo.com, merrysoares90@gmail.com, and afjdpguterres@gmail.com. Joshua R. Francis, Menzies School of Health Research, Global and Tropical Health, Casuarina, Australia, and Royal Darwin Hospital, Paediatrics, Casuarina, Australia, E-mail: josh.francis@menzies.edu.au.

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